Our principal object is the continuation of the investigation of the physiological role of the endogenous opioid peptides by correlating the results obtained from the binding at the three major mu, delta-, and kappa-sites with those obtained in pharmacological experiments. One of the important problems is the coupling mechanism between binding of an opioid ligand and the response of the effector system. In this respect, the investigation of the differences found in the binding of selective ligands at mu-, delta-, and kappa-sites and their modulation by monovalent and divalent cations and by guanyl nucleotides will be continued. The selective agonists are the mu-ligand (3H)-(D-Ala2,MePhe4,Gly-ol5)enkephalin, the delta-ligand (3H)-(D-Pen2,D-Pen5)enkephalin and the kappa- ligands (3H)-dynorphin A (1-9) and (3H)-U-69,593. It is of particular importance to extend this investigation to use the selective mu-antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP; V.J. Hruby), the selective delta-antagonist naltrindole and the selective kappa-antagonist norbinaltorphimine, both developed in the laboratory of Portoghese. The other important problem is the mechanism involved in the release of fragments of pro-enkephalin and Pro-dynorphin. The basis for the investigation of the release is published in J. Neurochemistry (in press). The principle of the method is the use of two successive HPLC systems which separate the endogenous opioid peptides for subsequent assay in the mouse vas deferens. It is now possible to determine the evoked release of (Met)enkephalin, (Leu)enkephalin, (Met)enkephalyl-Arg-Gly-Leu, and (Met)enkephalyl- Arg, Phe. BAM 8 amide, (Met)enkephalyl-Arg-Arg-Val-NH;, is also released but there is no detectable release of BAM 18 although it is present in the non-stimulated tissue. The amount of released opioid varies between 18 to 30% of the tissue content.